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Tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. An acute increase in tumor oxygenation before radiation treatment should therefore significantly improve the tumor cell kill after radiation. Efforts to increase oxygen delivery to the tumor have not shown positive clinical results. Here we show that targeting mitochondrial respiration results in a significant reduction of the tumor cells’ demand for oxygen, leading to increased tumor oxygenation and radiation response. We identified an activity of the FDA-approved drug papaverine as an inhibitor of mitochondrial complex I. We also provide genetic evidence that papaverine’s complex I inhibition is directly responsible for increased oxygenation and enhanced radiation response. Furthermore, we describe derivatives of papaverine that have the potential to become clinical radiosensitizers with potentially fewer side effects. Importantly, this radiosensitizing strategy will not sensitize well-oxygenated normal tissue, thereby increasing the therapeutic index of radiotherapy.

The unique metabolism of tumors was described many years ago by Otto Warburg, who identified tumor cells with increased glycolysis and decreased mitochondrial activity. However, \"aerobic glycolysis\" generates fewer ATP per glucose molecule than mitochondrial oxidative phosphorylation, so in terms of energy production, it is unclear how increasing a less efficient process provides tumors with a growth advantage. We carried out a screen for loss of genetic elements in pancreatic tumor cells that accelerated their growth as tumors, and identified mitochondrial ribosomal protein L28 (MRPL28). Knockdown of MRPL28 in these cells decreased mitochondrial activity, and increased glycolysis, but paradoxically, decreased cellular growth in vitro. Following Warburg's observations, this mutation causes decreased mitochondrial function, compensatory increase in glycolysis and accelerated growth in vivo. Likewise, knockdown of either mitochondrial ribosomal protein L12 (MRPL12) or cytochrome oxidase had a similar effect. Conversely, expression of the mitochondrial uncoupling protein 1 (UCP1) increased oxygen consumption and decreased tumor growth. Finally, treatment of tumor bearing animals with dichloroacetate (DCA) increased pyruvate consumption in the mitochondria, increased total oxygen consumption, increased tumor hypoxia and slowed tumor growth. We interpret these findings to show that non-oncogenic genetic changes that alter mitochondrial metabolism can regulate tumor growth through modulation of the consumption of oxygen, which appears to be a rate limiting substrate for tumor proliferation.

It has been known for many years that cellular metabolism within the solid tumour is markedly different from that of the corresponding normal tissue. The transcription factor hypoxia-inducible factor 1 (HIF1) has been implicated in regulating many of the genes that are responsible for the metabolic difference. However, it remains unclear how this 'aerobic glycolysis', originally described by Otto Warburg, offers tumour cells a growth advantage. As discussed in this Perspective, new data suggests that this metabolic switch may provide a benefit to the tumour not by increasing glycolysis but by decreasing mitochondrial activity. Aerobic glycolysis, or the 'Warburg effect', is a metabolic switch unique to tumour cells. But how might this change in tumour cell metabolism confer a growth advantage?

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro . Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.

Solid tumors frequently contain large regions with low oxygen concentrations (hypoxia). The hypoxic microenvironment induces adaptive changes to tumor cell metabolism, and this alteration can further distort the local microenvironment. The net result of these tumor-specific changes is a microenvironment that inhibits many standard cytotoxic anticancer therapies and predicts for a poor clinical outcome. Pharmacologic targeting of the unique metabolism of solid tumors could alter the tumor microenvironment to provide more favorable conditions for anti-tumor therapy. Here, we describe a strategy in which the mitochondrial metabolism of tumor cells is increased by pharmacologic inhibition of hypoxia-inducible factor 1 (HIF1) or its target gene pyruvate dehydrogenase kinase 1 (PDK1). This acute increase in oxygen consumption leads to a corresponding decrease in tumor oxygenation. Whereas decreased oxygenation could reduce the effectiveness of some traditional therapies, we show that it dramatically increases the effectiveness of a hypoxia-specific cytotoxin. This treatment strategy should provide a high degree of tumor specificity for increasing the effectiveness of hypoxic cytotoxins, as it depends on the activation of HIF1 and the presence of hypoxia, conditions that are present only in the tumor, and not the normal tissue.

Background Pyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1–4) and two reactivating phosphatases (PDP1, 2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth. Methods To isolate the function of the serine-dependent regulation of PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293, and 300 or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice. Results In this family of cells with non-phosphorylatable PDHA1, we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice. Conclusions We found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had a very modest reduction of growth in vitro, but failed to grow as tumors indicating that dynamic PDC adaptation to microenvironmental conditions is necessary to support pancreatic cancer growth in vivo.

Clinical evidence shows that tumor hypoxia is an independent prognostic indicator of poor patient outcome. Hypoxic tumors have altered physiologic processes, including increased regions of angiogenesis, increased local invasion, increased distant metastasis and altered apoptotic programs. Since hypoxia is a potent controller of gene expression, identifying hypoxia-regulated genes is a means to investigate the molecular response to hypoxic stress. Traditional experimental approaches have identified physiologic changes in hypoxic cells. Recent studies have identified hypoxia-responsive genes that may define the mechanism(s) underlying these physiologic changes. For example, the regulation of glycolytic genes by hypoxia can explain some characteristics of the Warburg effect. The converse of this logic is also true. By identifying new classes of hypoxia-regulated gene(s), we can infer the physiologic pressures that require the induction of these genes and their protein products. Furthermore, these physiologically driven hypoxic gene expression changes give us insight as to the poor outcome of patients with hypoxic tumors. Approximately 1–1.5% of the genome is transcriptionally responsive to hypoxia. However, there is significant heterogeneity in the transcriptional response to hypoxia between different cell types. Moreover, the coordinated change in the expression of families of genes supports the model of physiologic pressure leading to expression changes. Understanding the evolutionary pressure to develop a ‘hypoxic response’ provides a framework to investigate the biology of the hypoxic tumor microenvironment.

Human osteopontin (OPN), a known tumor associated protein, exists in different isoforms, whose function is unclear. It also possesses a RGD domain, which has been implicated in diverse function. Here, we use genetic approaches to systematically investigate the function of the RGD domain in different OPN isoforms on tumor progression and metastasis for 2 different solid tumor models. Using isoform-specific qRT-PCR, we found that OPN-A and B were the main isoforms overexpressed in evaluated human tumors, which included 4 soft tissue sarcomas, 24 lung and 30 head and neck carcinomas. Overexpression of either OPN-A or B in two different cell types promoted local tumor growth and lung metastasis in SCID mouse xenografts. However, expression of either isoform with the RGD domain either mutated or deleted decreased tumor growth and metastasis, and resulted in increased apoptosis by TUNEL staining. In vitro, whereas mutation of the RGD domain did not affect cell-cell adhesion, soft agar growth or cell migration, it increased apoptosis under hypoxia and serum starvation. This effect could be mitigated when the RGD mutant cells were treated with condition media containing WT OPN. Mechanistically, the RGD region of OPN inhibited apoptosis by inducing NF-kappaB activation and FAK phosphorylation. Inhibition of NF-kappaB (by siRNA to the p65 subunit) or FAK activation (by a inhibitor) significantly increased apoptosis under hypoxia in WT OPN cells, but not in RGD mutant cells. Unlike prior reports, our data suggest that the RGD domain of both OPN-A and B promote tumor growth and metastasis mainly by protecting cells against apoptosis under stressed conditions and not via migration or invasion. Future inhibitors directed against OPN should target multiple isoforms and should inhibit cell survival mechanisms that involve the RGD domain, FAK phosphorylation and NF-kappaB activation.

Hypoxia Inducible Lipid Droplet Associated (HILPDA) is frequently overexpressed in tumors and promotes neutral lipid storage. The impact of Hilpda on pancreatic ductal adenocarcinoma (PDAC) tumor growth is not known. In order to evaluate Hilpda-dependent lipid storage mechanisms, expression of Hilpda in murine pancreatic cells (KPC) was genetically manipulated. Lipid droplet (LD) abundance and triglyceride content in vitro were measured, and model tumor growth in nu/nu mice was determined. The results showed that excess lipid supply increased triglyceride storage and LD formation in KPC cells in a HILPDA-dependent manner. Contrary to published results, inhibition of Adipose Triglyceride Lipase (ATGL) did not ameliorate the triglyceride abundance differences between Hilpda WT and KO cells. Hilpda ablation significantly decreased the growth rate of model tumors in immunocompromised mice. In conclusion, Hilpda is a positive regulator of triglyceride storage and lipid droplet formation in murine pancreatic cancer cells in vitro and lipid accumulation and tumor growth in vivo. Our data suggest that deregulated ATGL is not responsible for the absence of LDs in KO cells in this context.

Although the prognosis for patients with glioblastoma is poor, survival is variable, with some patients surviving longer than others. For this reason, there has been longstanding interest in the identification of prognostic markers for glioblastoma. We hypothesized that specific histologic features known to correlate with malignancy most likely express molecules that are directly related to the aggressive behavior of these tumors. We further hypothesized that such molecules could be used as biomarkers to predict behavior in a manner that might add prognostic power to sole histologic observation of the feature. We reasoned that perinecrotic tumor cell palisading, which denotes the most aggressive forms of malignant gliomas, would be a striking histologic feature on which to test this hypothesis. We therefore used laser capture microdissection and oligonucleotide arrays to detect molecules differentially expressed in perinecrotic palisades. A set of RNAs (including POFUT2, PTDSR, PLOD2, ATF5, and HK2) that were differentially expressed in 3 initially studied, microdissected glioblastomas also provided prognostic information in an independent set of 28 glioblastomas that did not all have perinecrotic palisades. On validation in a second, larger independent series, this approach could be applied to other human glioma types to derive tissue biomarkers that could offer ancillary prognostic and predictive information alongside standard histopathologic examination.